RNA splicing factor RBFOX2 is a key factor in the progression of cancer and cardiomyopathy

Abstract Background Alternative splicing of pre‐mRNA is a fundamental regulatory process in multicellular eukaryotes, significantly contributing to the diversification of the human proteome. RNA‐binding fox‐1 homologue 2 (RBFOX2), a member of the evolutionarily conserved RBFOX family, has emerged as a critical splicing regulator, playing a pivotal role in the alternative splicing of pre‐mRNA. This review provides a comprehensive analysis of RBFOX2, elucidating its splicing activity through direct and indirect binding mechanisms. RBFOX2 exerts substantial influence over the alternative splicing of numerous transcripts, thereby shaping essential cellular processes such as differentiation and development. Main body of the abstract Dysregulation of RBFOX2‐mediated alternative splicing has been closely linked to a spectrum of cardiovascular diseases and malignant tumours, underscoring its potential as a therapeutic target. Despite significant progress, current research faces notable challenges. The complete structural characterisation of RBFOX2 remains elusive, limiting in‐depth exploration beyond its RNA‐recognition motif. Furthermore, the scarcity of studies focusing on RBFOX2‐targeting drugs poses a hindrance to translating research findings into clinical applications. Conclusion This review critically assesses the existing body of knowledge on RBFOX2, highlighting research gaps and limitations. By delineating these areas, this analysis not only serves as a foundational reference for future studies but also provides strategic insights for bridging these gaps. Addressing these challenges will be instrumental in unlocking the full therapeutic potential of RBFOX2, paving the way for innovative and effective treatments in various diseases.


INTRODUCTION
RNA-binding proteins (RBPs) play pivotal roles in orchestrating RNA-protein interactions, governing all facets of RNA metabolism from its inception to its degradation. 1hese multifaceted proteins harbour diverse RNA-binding domains (RBDs), enabling them to recognise a spectrum of short RNA elements and auxiliary non-RBDs. 2RBPs exhibit the capability to identify and interact with particular RNA sequences via their respective RBDs.This interaction facilitates a spectrum of crucial cellular processes, encompassing alternative splicing, polyadenylation, nuclear export, intracellular localisation, degradation and regulation of translation. 3ithin the lineage of evolutionarily conserved RBPs, the RBFOX family emerges as a pivotal regulator, particularly notable for its role in modulating alternative pre-mRNA splicing.This regulatory function is especially pronounced in the developmental contexts of brain, heart and muscle tissue. 4The RBFOX family has been elucidated as a central player in orchestrating the alternative splicing program of numerous genes, underscoring its pivotal role in mediating tissue-specific regulatory processes. 5The gene for one prominent member of this family, RNA-binding fox-1 homologue 2 (RBFOX2), alternatively known as RNA-binding motif protein 9 (RBM9), is situated on the negative strand of chromosome 22q12.3(hg38 chr22:35738736-36028824), encompassing a total gene length of 290 089 bp.Mirroring the behaviour of other RBFOX proteins, RBFOX2 predominantly influences spliced isoform expression through its modulation of alternative splicing events in target gene exons. 4Recent investigations have unveiled RBFOX2's pivotal regulatory roles across multiple biological processes, including cell proliferation, 6 invasion, migration 7 and excitation-contraction (E-C) coupling. 8erturbed RBFOX2 expression has been correlated with the onset and progression of various malignancies 9 and cardiovascular disorders. 10Simultaneously, aberrations in RBFOX2 have been implicated in conferring resistance to anticancer drugs, such as tamoxifen (TAM) 11 and Rebecsinib. 6s a central player in the realm of splicing regulation, a potential biomarker and a therapeutic target for a multitude of disease-associated genes, RBFOX2 holds substantial promise for research endeavors.While studies pertaining to RBFOX2 are steadily on the rise, there is a conspicuous dearth of comprehensive reviews elucidating its molecular regulatory mechanisms and proposing targeted therapeutic strategies under both physiological and pathological conditions.Consequently, this article aims to systematically synthesise knowledge about RBFOX2, pinpoint the advancements and lacunae in current research, and furnish a directional compass and theoretical framework for ensuing investigations in this field.

ELUCIDATING THE PROTEIN DOMAIN ARCHITECTURE AND TRANSCRIPT PROFILE OF RBFOX2
RBFOX constitutes a class of alternative splicing regulators that have endured through mammalian evolution. 5Currently, researchers have identified three proteins within the RBFOX family: RBFOX1, RBFOX2 and RBFOX3. 12All members of the RBFOX family share a relatively conserved RNA-recognition motif (RRM), which exhibits a highaffinity binding capacity for the hexanucleotide sequence (U)GCAUG (Figure 1). 12,13These RRMs are indispensable for the specific recognition of RNA sequences by RBFOX family proteins. 12Simultaneously, beyond its recognition of the (U)GCAUG sequence, the RRM of RBFOX has demonstrated an additional capacity to bind to the GCACG sequence.However, it is noteworthy that its binding affinity towards GCACG is comparatively diminished when juxtaposed with (U)GCAUG. 14Intriguingly, the functionality of RBFOX proteins extends beyond this motif, as they can be indirectly recruited to RNA through participation in the formation of larger assemblies known as large assembly of splicing regulator (LASR) complexes, and can even exhibit splicing activity in the absence of the (U)GCAUG motif. 15urthermore, within the RBFOX family, it has been observed that both RBFOX1 and RBFOX2 proteins possess a conserved nuclear localisation signal (NLS) situated in the C-terminal domain (CTD). 13This NLS plays a pivotal role in ensuring strong nuclear localisation of RBFOX family proteins under non-stress conditions. 17Under stress conditions, RBFOX family proteins show cytoplasmic localisation mediated by stress granules. 18When cells are under stress, numerous proteins and RNAs aggregate in the cytoplasm to form non-enveloped structures known as stress granules. 19RBFOX family proteins strongly bind to stress granules through their RRM, thereby shifting from nuclear to cytoplasmic localisation. 18Alterations in the subcellular localisation of these proteins are intimately connected to changes in their capacity to regulate alternative splicing. 20It is worth noting that aside from the RRM and NLS regions, the remaining sequences of RBFOX proteins display limited conservation. 13n addition to their structural features, RBFOX family coding genes can generate a multitude of distinct transcripts through alternative splicing of RRM coding domains and some exons, which affects their subcellular localisation and regulatory functions in alternative splicing. 21RBFOX2 comprises 14 exons and 13 introns, F I G U R E 1 Decoding RNA-binding fox-1 homologue 2 (RBFOX2): insights into structure, function and RNA-binding conformation.(A) Primary structure of RBFOX2: the protein sequence of RBFOX2 is compared with its family proteins RBFOX1 and RBFOX3.Notably, the sequences of the RNA-recognition motif (RRM) and nuclear localisation signal (NLS) are identical in RBFOX2 and RBFOX1.(B) Comparison of RRM between RBFOX2 and RBFOX1: a detailed comparison of the RRM between RBFOX2 and RBFOX1 highlights their structural similarities.(C) RNA-binding conformation of RBFOX1 with UGCAUGU motif: the RNA-binding conformation of RBFOX1 with the UGCAUGU motif is depicted.Within this motif, Phe126 plays a crucial role in binding U1, G2, C3 and A4, while His120, Phe158 and Phe160 are essential for binding U5, G6 and U7.Protein structure data utilised in this analysis is sourced from the Protein Data Bank (2CQ3 and 2ERR) and visualised using ChimeraX. 16(D) Various transcripts of human RBFOX2 depict a complex exon organisation, where constitutive exons are delineated by orange boxes, alternative exons by grey-blue boxes, and regions potentially untranslated by red gradients.Exon numbering serves for precise identification, correlating with genomic coordinates detailed in Table S1.We numbered the RBFOX2 exons with Arabic numerals based on the order of their starting sites on the chromosome.Alternative exons were numbered with letters according to their starting sites on the chromosome.This splicing procedure ensures that each exon has a unique identifier, allowing for a clearer description of the gene structure and alternative splicing forms.It is important to note that this exon numbering may not align with previously reported exon designations (coordinate information of RBFOX2 exons based on GRCh38/hg38).
generating a total of 20 transcripts, 15 of which have protein-coding potential.Different transcripts can be translated to produce protein isoforms with varying splicing activities.For example, exon 6 encodes 31 of the 73 residues of the RBFOX2 RRM. 22Skipping exon 6 eliminates β-strand 3 and α-helix 2 from the RBFOX2 structure.β-Strand 3 contains the key RNP1 motif for RRM folding and has several crucial RNA contact sites. 12Thus, exon 6 is key to the structural integrity of RBFOX2 RRM, and its omission will completely alter the protein's conformation and RNA-binding ability.In-depth study of the RBFOX2-ΔRRM isoform revealed that RBFOX2-ΔRRM not only lost its RNA binding activity but also inhibited the alternative splicing function of other RBFOX2 proteins containing a complete RRM.Therefore, scientists classified these RBFOX2 isoforms that act in a dominant negative (DN) manner as DN subtypes. 22Different transcripts also produce protein isoforms with different subcellular localisations.For example, exon 9 contains a 5-amino acid coding sequence (Ser-Leu-Pro-Leu-Val) associated with RBFOX2 subcellular localisation.By including this peptide, RBFOX2 can translocate from the nucleus to the cytoplasm. 23Deletion of RBFOX2 exon 10 likely causes a frameshift, resulting in the RBFOX2-ΔE10 isoform, which contains a PTEVT amino acid sequence at the C-terminus, causing migration to the cytoplasm. 24,25n addition, different transcripts of RBFOX2 are closely related to its indirect binding mode mediated by the LASR complex, which can further affect the splicing activity of RBFOX2.For example, a pair of 43 and 40 nt exons in the CTD coding sequence are specific for encoding muscle (RBFOX2-43) and non-muscle isoforms (RBFOX2-40) of RBFOX2, respectively.The 40 nt exon-encoded peptide chain contains three tyrosine residues, which are crucial for RBFOX2 to participate in the assembly of LASR. 26,27owever, muscle isoforms (RBFOX2-43) lack these tyrosine residues and cannot function through the indirect binding mode, resulting in limited splicing activity. 26istinguished from other RBFOX family proteins, RBFOX2's unique CTD orchestrates the recruitment of the polycomb repressive complex 2 (PRC2) to target genes. 28BFOX2 also acts as a reader recognising specific epigenetic marks on RNA, such as N6-methyladenosine (m6A) 29 and 5-hydroxymethylcytosine (hm5C). 30The PRC2-mediated histone modification (H3K27me3) plays a crucial role in the aberrant silencing of downstream genes 28 and is a feature observed across diverse cell types in mammals. 31When RBFOX2 engages with chromatinassociated RNAs (caRNAs), it acts as a mediator for the recruitment of the PRC2 complex.Consequently, the PRC2 complex induces histone methylation within the gene promoter region, effectively inhibiting transcription. 28In a recent breakthrough, researchers discovered that RBFOX2 recognises m6A modification on caRNAs. 29RBFOX2 demonstrates a specific ability to recognise and bind m6A modifications on caRNAs, precisely by virtue of its RRM's recognition of the AGm6AUG sequence. 29This event subsequently prompts RBFOX2 to enlist RBM15, a constituent of the methyltransferase complex, to modify promoter-associated RNAs with m6A.The interaction between RBM15 and the m6A reader YTHDC1 facilitates the recruitment of YTHDC to the site where RBFOX2 binds.Ultimately, YTHDC ushers in the PRC2 complex to the same locus as RBFOX2, thereby instigating H3K27me3 histone modifications at neighboring promoter sites, culminating in transcriptional repression. 28,29,32When RBFOX2 acts as a reader of m6A, it plays an important role in acute myeloid leukaemia (AML).Overexpressed RBFOX2 inhibits the expression of TGFB1 through the RBFOX2/m6A/RBM15/YTHDC1/PRC2 axis, blocks the transforming growth factor-beta (TGF-β) signalling pathway, and promotes the progression of AML.Downregulation of RBFOX2 significantly inhibits the survival and proliferation of AML cells and promotes myeloid differentiation.RBFOX2 is essential for the self-renewal and stemness maintenance of AML cells, making it a potential target for leukaemia treatment. 29Additionally, Dou et al. found that RBFOX2 can act as a reader of RNA hm5C, exerting specific recognition and degradation functions on target genes.Specifically, the methyltransferase NSUN5 first acts as a writer to introduce m5C modification on the caRNA of CTNNB1, which encodes β-catenin.NSUN5 then recruits TET2 and RBFOX2 to chromatin.Under the action of TET2, m5C on caRNA is converted to hm5C.Wu et al.'s study first discovered that RBFOX2 can act as a reader of hm5C to inhibit the expression of CTNNB1.However, the specific molecular mechanism by which RBFOX2 inhibits the expression of CTNNB1 has not yet been explored.One possibility is that, similar to its role as a reader of m6A, RBFOX2 regulates the expression of target genes by recruiting the PRC2 complex.This requires further in-depth research. 30This synergistic function of RBFOX2 complements its role in transcriptional repression by recruiting PRC2, indicating that RBFOX2 can act as a multifunctional protein coordinating its interaction with multiple targets. 30

UNRAVELING THE VERSATILE FUNCTIONS AND MODES OF ACTION OF RBFOX2 IN ALTERNATIVE SPLICING REGULATION
Alternative splicing is a pivotal determinant of the intricate complexity of multicellular eukaryotic transcriptomes.Its global and gene-specific regulation governs a wide spectrum of processes, encompassing tissue specificity, cell differentiation and the onset of cancer. 33Among the family of RBFOX proteins, RBFOX2, akin to its counterparts, plays a central role primarily within the nucleus as a splicing regulator. 34s detailed in Table 1 and illustrated in Figure 2, recent studies conducted over the past few years have revealed RBFOX2's exceptional capacity to discern unique alternatively spliced RNAs across various tissues.24,35 Leveraging its intrinsic RRM and its specific binding to the (U)GCAUG sequence, RBFOX2 can selectively engage target RNA molecules.12 Interestingly, not all RBFOX2 splicing targets adhere to the (U)GCAUG motif.28 In instances where a complete (U)GCAUG sequence is absent, RBFOX2 employs an indirect approach by recruiting other RBPs to execute splicing functions.15,36 Thus, the functions of RBFOX2 are categorised into two distinct modes based on its binding strategies: direct binding modes requiring the (U)GCAUG recognition sequence, 13 and indirect binding modes that rely on the recruitment by additional proteins.15,36 In the direct binding mode, RBFOX2 exhibits a remarkable ability to specifically recognise and bind both primary splicing factor binding sequences (GCAUG) and secondary splicing factor binding sequences (GCCUG or GCUUG) through its conserved RRM, subsequently localising to the target splicing site.36,51 RBFOX2 typically targets introns in precursor mRNA (pre-mRNA), 50 3′ untranslated regions (UTRs) in mRNA, 54 and even pre-miRNA.55 Notably, when RBFOX2 binds downstream binding sites of a target exon, exon inclusion is typically promoted, whereas binding upstream binding sites of a target exon inhibits exon inclusion.6 Due to the variable promoter and alternative splicing events that can diversify RBFOX2 gene products, the structure of RBFOX2's RRM is particularly susceptible to disruption.Consequently, RBFOX2 subtypes can be classified into RBFOX2-wild type (WT) with an intact RRM and RBFOX2-DN subtypes lacking a complete RRM. 22 Dyregulation of any RBFOX2 subtype in humans can lead to aberrant splicing in related genes and subsequent disease manifestations.7,56 Notably, the isoforms of the RBFOX gene can also be modulated by In the direct binding mode, RBFOX2 specifically recognises target RNA sequences containing the (U)GCAUG motif through its inherently conserved RNA-recognition motif (RRM).When RBFOX2 binds upstream of a target exon splice site, it consistently leads to exon skipping.Conversely, binding downstream of the splice site results in exon inclusion.In the indirect binding mode, RBFOX2 interacts with target RNA indirectly by binding to other RNA-binding proteins (RBPs).This interaction enables RBFOX2 to cross-link with the target RNA, allowing it to exert splicing activity regardless of whether the RNA sequence contains the complete (U)GCAUG motif or not. PB, protein-binding domain; RBD, RNA-binding domain; RBP, RNA-binding protein; RRM, RNA-recognition motif.Created with BioRender.com.
other RBPs; for instance, overexpression of CELF1 triggers a transition from the RBFOX2-WT isoform to the RBFOX2-40 isoform (an RBFOX2-DN isoform). 26Certain subtypes of other RBFOX family members have been observed to influence RBFOX2's RRM, giving rise to RBFOX2-DN subtypes. 22,57In mice, for instance, the Rbfox3 can impede exon 6 inclusion in Rbfox2 pre-mRNA, causing an inframe deletion of RRM and the consequent emergence of the Rbfox2-DN isoform. 22Certain sno-lncRNAs, such as SNORD116, exhibit the ability to recruit and form complexes with RBFOX2.This interaction impedes the nuclear migration of RBFOX2, consequently attenuating its role in modulating alternative splicing within the nucleus. 58n the indirect binding mode, RBFOX2 forms associations with target RNA indirectly through interactions with other RBPs. 36For example, RBFOX2 can be recruited by SRSF1, which binds within or downstream of a target exon, thereby participating in alternative splicing without directly engaging the target RNA. 36Similarly, the RBPs recruiting RBFOX2 for indirect RNA binding may also comprise one or more components of a protein complex.Notably, hnRNP M within the LASR complex, a splicing complex, has been identified as an RBFOX2 recruiter, enhancing RBFOX2's binding to target genes. 36RBFOX2 can be indirectly recruited to cross-linking sites by directly binding to hnRNP M as part of the LASR complex, 15 allowing it to cross-link with target RNA with or without a complete (U)GCAUG motif and to facilitate splicing activity. 15ecent studies have even unveiled RBFOX2's recruitment by the smooth muscle-specific regulator RBPMS, forming a distinct, cell-specific splicing regulatory complex distinct from LASR. 59ecent studies have shown that in addition to producing different mRNA transcripts through alternative splicing, another outcome of RBFOX2 splicing is the induction of nonsense-mediated RNA decay (NMD).NMD is an mRNA surveillance pathway that recognises and selectively degrades transcripts containing premature termination codons (PTCs), preventing the production of incomplete or abnormal proteins. 60RBFOX2 can introduce PTCs into its downstream mRNA through alternative splicing, causing these mRNAs to be degraded via the NMD pathway.This gene regulation mode, where alternative splicing is coupled with NMD, is also called AS-NMD. 61NMD plays an important role in the expression of RBPs. 62As shown in study by Jangi et al., RBPs are particularly enriched in genes regulated by RBFOX2 through AS-NMD. 61These RBPs regulated by RBFOX2 greatly expand its regulatory network.Additionally, since RBFOX2 itself is also an RBP, its mRNA may also be affected by other RBPs in the cell and undergo NMD.This forms a cross-regulatory competition mechanism between RBFOX2 and other RBPs, wherein they influence each other's expression levels and competitively regulate the expression of downstream genes.This regulatory mechanism increases the complexity of gene expression regulation in cells and may help cells adapt to environmental changes or perform specific functions. 61,63

DECIPHERING THE INTRICATE REGULATION OF RBFOX2 SUBCELLULAR LOCALISATION
RBFOX1 and RBFOX2 proteins consistently demonstrate strong nuclear localisation under non-stressed conditions. 17,21This localisation pattern is influenced by alternative splicing events involving the coding sequence of their CTD, giving rise to nuclear localisation isoforms containing NLS and cytoplasmic localisation isoforms devoid of NLS. 4 However, in contrast to RBFOX1 proteins, the nuclear localisation of RBFOX2 is subject to additional, more stringent regulatory mechanisms beyond its conserved C-terminal NLS. 25 RBFOX2 undergoes transcription from at least four distinct promoters, generating an array of isoforms with diverse properties. 64Some RBFOX2 isoforms have unique N-terminal domains (NTD). 25 When a functional NLS is generated within this unique NTD, it can enable certain subtypes to exhibit nuclear localisation in the absence of a C-terminal NLS. 25 For example, in mouse mammary epithelial cells, there are two Rbfox2 isoforms with different NTDs, Rbfox2-1A and Rbfox2-1F. 65Both isoforms skip or splice exon B40, resulting in frameshifts and the absence of the typical C-terminal NLS.The NTD of Rbfox2-1A contains a potential amino acid sequence that functions as a functional NLS, allowing Rbfox2-1A to show nuclear localisation, unlike Rbfox2-1F, which shows cytoplasmic localisation. 25urthermore, the subcellular localisation of RBFOX2 can also be influenced by isoforms of other RBFOX family members.For instance, during early chick embryo development, the Rbfox3-Δ31 isoform, characterised by the exclusion of exon 31aa, is notably abundant in the cytoplasm.This specific isoform, Rbfox3-Δ31, possesses the ability to modulate the splicing activity of Rbfox2 by altering its subcellular localisation. 34ince post-transcriptional regulation steps usually occur in specific subcellular compartments, such as the nucleus, mitochondria and Golgi apparatus, 66 abnormal subcellular localisation can theoretically affect the activity of RBFOX2 as a splicing factor in the nucleus, potentially leading to pathological changes. 25Under normal nuclear localisation, RBFOX2 regulates RNA splicing by binding to pre-mRNA, essential for developing and maintaining tissues such as the brain, 35 muscle 67 and heart. 68The translocation of RBFOX2 to the cytoplasm may regulate its activity as a splicing factor in the nucleus, closely linked to neuronal degeneration, 69 muscle atrophy 70 and congenital heart disease. 54This translocation is also associated with various malignant phenotypes of tumour cells, such as enhanced cell proliferation, inhibited apoptosis, invasion and migration. 25,51

RBFOX2 AND CARDIOVASCULAR DISEASE AND HEALTH
Cardiovascular disease (CVD) stands as a formidable global health challenge, encompassing a spectrum of conditions including cardiomyopathy, coronary heart disease (CHD), hypertension, heart valve disease, arrhythmia, etc. 71 Recent research endeavours have illuminated RBFOX2's potential utility as a diagnostic and prognostic biomarker for CVDs, with mounting evidence linking its dysregulated expression to the onset of these diseases.RBFOX2, through its intricate molecular mechanisms, contributes significantly to the maintenance of myocardial development and function, playing a pivotal role in the physiological and pathological processes that underpin the cardiovascular system.

RBFOX2 dysregulation in CVDs
The expression profile of RBFOX2 exhibits significant dysregulation across a spectrum of CVDs, as summarised in Table 2. Downregulated RBFOX2 expression characterises conditions, such as dilated cardiomyopathy (DCM) 68 and CHD. 41,72Conversely, diseases marked by elevated RBFOX2 expression include arrhythmia, 26 myotonic dystrophy type 1 (DM1) cardiomyopathy 26 and hypertension. 37owever, the expression of RBFOX2 in diabetic heart disease (DHD) 56,73 remains subject to controversy.In a CHD model established using streptozotocin, Rbfox2 expression is significantly reduced in whole heart tissues. 41Similar observations are made in cardiovascular tissues from CHD patients, where RBFOX2 expression is markedly decreased compared to normal samples. 72Wei et al. deleted the Rbfox2 gene by crossing 129S6/SVEvTac mice, with LoxP sites flanking Rbfox2 exons 6 and 7, with Mlc2v-Cre transgenic mice, using the Cre-LoxP recombinase system.Compared to normal mice, the heart sections of Rbfox2 conditional deletion mice exhibited significant ventricular enlargement and thinning of the ventricular wall, ultimately developing into typical DCM.Notably, their results confirmed that Rbfox2 deletion leads to DCM, which subsequently causes heart failure, indicating that downregulation of Rbfox2 is not merely a functional consequence of heart failure. 68otably, the upregulation of RBFOX2 is often associated with a specific isoform, the RBFOX2-DN isoform.In DHD, RBFOX2 expression is elevated in left ventricular cardiomyocytes of human patients with type 2 diabetes mellitus, and this upregulation coincides with increased expression of RBFOX2-DN isoforms. 73Similarly, Rbfox2 expression is increased in rat vascular smooth muscle cells (VSMCs) and upregulated in hypertensive arteries, accompanied by enhanced expression of DN subtypes. 37In heart samples from patients with DM1 cardiomyopathy or DM1 non-diabetic arrhythmia, RBFOX2 upregulation is coupled with the selective upregulation of the RBFOX2-40 isoform (a type of RBFOX2-DN). 26owever, paradoxically, a recent study reveals that Rbfox2 expression is downregulated in the heart tissue of diabetic rats induced by high-fat feeding compared to the control group. 56Despite this downregulation, the levels of Rbfox2-DN exhibit a clear upward trend, aligning with previous findings. 73This suggests that upregulation of RBFOX2-DN isoforms is a consistent phenomenon in diabetic hearts.Nonetheless, the overall up-or downregulation of RBFOX2 levels may be governed by changes in other isoforms, influenced by distinct disease states.
Furthermore, the subcellular localisation of RBFOX2 varies within different cell types.In isolated rat VSMCs and rat mesenteric artery smooth muscle cells, Rbfox2 predominantly localises to the nucleus. 37In contrast, in hypoplastic left heart syndrome (HLHS), RBFOX2 is primarily cytoplasmic in the hearts of HLHS patients. 54Thus, the ectopic expression of RBFOX2 emerges as a hallmark feature of cardiovascular cytopathies.

RBFOX2 affects cardiovascular health and disease
Cardiac dysfunction constitutes a significant contributor to CVDs, encompassing a spectrum of issues ranging from Ltype calcium channel Ca V 1.2 function to defects in cardiac E-C coupling and mitochondrial integrity (Figure 3). 56,74n increasing body of research underscores the pivotal role of RBFOX2 as a key regulatory factor in myocardial E-C coupling, Cav1.2 channel function, and the maintenance of mitochondrial morphology.RBFOX2 emerges as a critical player in preserving normal myocardial development and overall cardiovascular health.
Cardiac E-C coupling orchestrates the transition from electrical excitation to muscle fibre contraction, underpinning proper cardiac function. 8While Jph2 variants have been linked to diverse cardiac disorders, the mechanisms underlying these associations have remained elusive. 75,76ecent findings in neonatal rat cardiomyocytes shed light on this conundrum, demonstrating that reduced Rbfox2 levels trigger the maturation of miR-34a.miR-34a, in turn, targets and inhibits the expression of Jph2, resulting in E-C coupling defects that culminate in impaired cardiac contraction and, ultimately, heart failure. 8,38he Cav1.2 channel, encoded by the CACNA1C gene, represents a critical substrate for cardiac electrophysiological activity. 77It participates in E-C coupling, modulates action potential duration, regulates cardiac cell gene expression and plays a central role in cardiac function. 78Aberrant Cav1.2 channel activity can induce premature depolarisation of cardiomyocytes, leading to severe arrhythmias and sudden cardiac death. 79In hypertensive rat VSMCs, elevated Rbfox2 inhibits the inclusion of Cacna1c exon 9′ and promotes the inclusion of exon 33.1][82] Furthermore, exon 9′ has been shown to play a pivotal role in the contraction of cerebral arteries. 83Consequently, dysregulation of Rbfox2's influence on Cav1.2 channel function emerges as a key contributor to hypertension pathogenesis. 37Cacna1c is the gene encoding Cav1.2, a critical subtype of the L-type calcium channel in cardiomyocytes.In diabetic rats, when RBFOX2 is downregulated by advanced glycation endproducts, it promotes the inclusion of Cacna1c exon 9′ and the expression of Cav1.2.Upon sarcolemma depolarisation, Ca 2+ enters the cell through membrane-bound Cav1.2, inducing calcium-induced calcium release from the sarcoplasmic reticulum into the cytoplasm.Downregulation of RBFOX2 leads to excessive Cav1.2 on the membrane, resulting in excessive Ca 2+ in the sarcoplasm, continuous contraction of muscle fibres, and subsequent myocardial hypertrophy in diabetic patients. 56itochondria are pivotal in sustaining cardiac energy metabolism, with preserving their intact morphology forming the bedrock of their functionality. 84Mitochondrial abnormalities lead to energy deficits in cardiac tissue, precipitating bioenergetic diseases such as heart failure. 85Research has elucidated that in H9c2 cells, the gene Slc25a4 possesses two distinct polyadenylation sites, resulting in the generation of mRNA molecules with differing lengths of 3′ UTR.Notably, the mRNA variant featuring an extended 3′ UTR exhibits diminished stability and translation efficiency, thereby impeding Slc25a4 expression.The protein Rbfox2 intervenes in this process by counteracting the influence of the distal polyadenylation site.Through its binding affinity to the UGCAUG motif downstream of the distal polyadenylation site on Slc25a4, Rbfox2 prompts the preferential production of mRNA with a shorter 3′ UTR.Diminishment of Rbfox2 levels results in an elevation of Slc25a4 mRNA harbour-ing the longer 3′ UTR, consequently dampening Slc25a4 expression.This dysregulation culminates in mitochondrial dysfunction within H9c2 cells, a phenomenon closely linked to cardiomyopathy pathogenesis. 10,86Moreover, during cardiac differentiation, RBFOX2 selectively promotes the inclusion of MICU1 exon 5′, endowing specificity to mitochondrial Ca 2+ uptake, a critical component for ATP production during muscle contraction. 39,87n addition, multiple splicing events coordinated by Rbfox2 contribute to myoblast fusion.For example, during myocyte differentiation, Rbfox2 promotes the production of two muscle-specific isoforms (Mef2d and Rock2) that synergistically control the terminal differentiation of myoblasts into multinucleated myotubes. 88,89n heart failure, reduced mouse Rbfox2 expression inhibits the inclusion of exon 13 in Enah and exon 4 in Sorbs2, while during heart development, 38 mouse Rbfox2 governs the balance by repressing Tpm1 isoforms containing exon 6a 40 and promoting Tpm1 isoforms with exon 9b. 10 Dysregulated splicing patterns of key genes involved in the cGMP-PKG-Ca 2+ pathway, such as Atp2b1 exon 21 and Mef2a exon 9, heighten the risk of cardiomyopathy and heart failure in patients with CHD and diabetes. 41n diabetic mouse foetal hearts, the inhibition of the PKC-mediated activation of Rbfox2 disrupts early developmental homeostasis. 90Additionally, RBFOX2 influences the alternative splicing of genes like MBNL1, MCAM, VCL and ACTN1, with downstream effects on the differentiation and function of cardiac smooth muscle cells. 91It is worth noting that the Gazzara et al. confirmed the splicing antagonism between CELF2 and the RBFOX family through knockdown and overexpression experiments in human cells, showing that CELF2 inhibits RBFOX2 mRNA and protein levels. 92Both CELF2 and RBFOX proteins are involved in the development and maintenance of neurons, muscle and cardiac tissues, and their antagonism contributes to splicing regulation in normal cardiac development and disease. 57,93However, the crosstalk between these proteins has not been directly studied.A deeper understanding of CELF2 and RBFOX2's functional antagonism may explain differences in expression and splicing between muscle and brain.
Given its pivotal role in cardiovascular health, the dysregulation of RBFOX2 can lead to a range of CVDs, including cardiomyopathy 10 and heart failure. 38Antagomir, a synthetic miRNA inhibitor capable of binding to specific miRNAs, has shown promise in ameliorating heart failure characterised by low Rbfox2 expression.Multiple upregulated cardiac key miRNAs (let-7f, miR-16, miR-200b) in heart failure negatively regulate Rbfox2.However, antagomir cocktail therapy has demonstrated the potential to restore Rbfox2 levels in a mouse cardiomyocyte-induced heart failure model. 38Consequently, antagomir presents a potential therapeutic avenue for treating heart failure with a low expression of RBFOX2 phenotype. 38

DYSREGULATED RBFOX2 IN CANCER PROGRESSION
Cancer stands as the foremost contributor to global mortality. 94Within the realm of cancer, RBFOX2 emerges as a potential biomarker, wielded for both diagnostic and prognostic purposes, while its aberrant expression forms an intricate association with the extent of tumour malignancy.Furthermore, RBFOX2 exhibits the capability to foster the phenotypic traits of cancer cells and steer the inexorable march of tumour progression.Notably, RBFOX2 plays a critical role in bolstering the viability of cancer cells even following treatment, thereby constituting a substantial contributing factor to the development of tumour resistance against anticancer therapies.

RBFOX2 dysregulation and subcellular localisation as diagnostic and prognostic indicators in cancer
In the context of cancer, RBFOX2 undergoes dysregulation at various levels within cells and tissues.Aberrant RBFOX2 expression serves as a valuable biomarker for assessing the malignancy of cancer.Additionally, abnormal subcellular localisation of RBFOX2 holds promise for evaluating the degree of malignancy.As presented in Table 3, cancer types exhibiting elevated RBFOX2 expression encompass lymphoma, 42 uveal melanoma (UM), 95 laryngeal cancer, 46 nasopharyngeal carcinoma (NPC), 32 gastric cancer (GC), 96 colorectal cancer (CRC) 97 and breast cancer (BrC). 7,98Conversely, cancers with reduced RBFOX2 expression include glioblastoma multiforme (GBM) 43 and pancreatic ductal adenocarcinoma (PDAC). 51n lymphoma, RBFOX2 expression significantly surpasses that in primary human T cells, as evidenced by its higher levels in human lymphoma cells (Karpas299 and Jurkat). 42Within the UM cohort, analysis of data from TCGA and ROMScohort reveals heightened RBFOX2 expression in patients at higher risk. 95Laryngeal carcinoma cells (Tu177, M4E, Hep2 and Tu212) show elevated RBFOX2 expression relative to normal nasopharyngeal epithelial cells (NP69) in laryngeal cancer. 46Furthermore, tumour tissues from NPC patients exhibit significantly higher RBFOX2 levels than mucosal tissues from rhinitis patients. 32GC cell lines (MCF-10ASGC7901, MGC803, BGC823 and AGS) manifest increased RBFOX2 expression compared to human normal gastric mucosal epithelial cells (GES1). 96In CRC, RBFOX2 registers upregulation in cancer tissues compared to normal counterparts. 97imilarly, in BrC, hypoxia-treated cell lines (MCF7 and HCC1806) display higher RBFOX2 expression than their normoxia-treated counterparts.In more malignant BrC patients, specifically those with triple-negative breast cancer (TNBC), RBFOX2 expression significantly exceeds that in other BrC patients, as indicated by TCGA data. 7,101n contrast, within the context of PDAC, patient-derived xenograft (PDX) models from metastatic PDAC display reduced RBFOX2 expression relative to PDX models derived from primary PDAC tumour tissue. 51Moreover, in glioma, RBFOX2 expression is lower in lesion brain tissue of glioma patients compared to normal brain tissue, and within glioma patients, those with higher malignancy exhibit lower RBFOX2 expression in lesion brain tissue than those with less malignancy. 43he abnormal expression pattern of RBFOX2 in AML remains uncertain.A study utilising the GEO database (GSE13159) revealed that AML patients across various subtypes, including normal karyotype, mixed lineage leukaemia and t(15:17), exhibited significantly elevated RBFOX2 expression in bone marrow samples compared to healthy counterparts.Additionally, Western blot analysis demonstrated higher RBFOX2 expression in five different types of AML cell lines (MM6, MOML13, NOMO1, NB4 and K562) compared to normal human myeloid cell line CD34 + HSCs/HPCs. 29However, contrasting findings emerged from studies employing fluorescence-activated cell sorting, indicating that paediatric AML-derived haematopoietic stem cells (HSCs) and haematopoietic progenitor cells (HPCs) expressed lower levels of RBFOX2 than their cord blood-derived counterparts. 6The observed differential expression patterns of RBFOX2 in paediatric AML and adult AML may stem from variances in the splicing events occurring within these distinct contexts. 6These conflicting results underscore the complexity of RBFOX2 expression regulation in AML and highlight the need for further research to elucidate its role in this context.
It is important to note that RBFOX2 predominantly localises within the nucleus, although its cytoplasmic localisation appears to be cancer cell-specific.For instance, in CRC, normal colon tissues exhibit nuclear RBFOX2 localisation, whereas strong cytoplasmic RBFOX2 staining is observed in various regions of CRC tissues.Quantitative assessment of nucleoplasmic distribution of RBFOX2 in cells further demonstrates significant differences between normal colon tissue and colon cancer tissue. 97urthermore, as delineated in Table 4, RBFOX2 emerges as an independent adverse prognostic factor in various cancer types, including AML, NPC, UM and GC.AML patients exhibiting high RBFOX2 expression demonstrate poorer overall survival. 29High RBFOX2 expression in NPC is associated with diminished overall survival and disease-free survival. 32In UM, elevated RBFOX2 expression correlates with worsened overall survival. 95In GC, heightened RBFOX2 expression links to advanced clinical and TNM stages, as well as inferior overall survival. 96,102Conversely, in other cancers such as PDAC, hepatocellular cancer (HCC) and GBM, the prognostic implications of RBFOX2 dysregulation contrast with the aforementioned findings.PDAC patients displaying low RBFOX2 expression experience inferior overall survival. 51Similarly, HCC patients exhibiting low RBFOX2 expression have a poorer overall survival, 49 and GBM patients with reduced RBFOX2 expression encounter diminished overall survival. 43

RBFOX2 drives tumour progression
The uncontrolled proliferation and metastasis of tumour cells within the human body represent the primary challenges in the quest to conquer cancer. 106In recent years, mounting evidence underscores the pivotal role of RBFOX2 in tumour progression.As depicted in Table 4 and Figure 4, RBFOX2 exhibits a nuanced influence on cancer, sometimes promoting tumour proliferation and metastasis, while in other instances, inhibiting these processes.BrC stands out as a highly heterogeneous tumour arising from mammary duct epithelial cells, distinguished by its robust invasive and metastatic potential. 107,108Within BrC cell lines (MCF-7, HCC-1806 and MDA-MB-231), RBFOX2 significantly bolsters the invasion and migration of cancer cells. 7,98,105In hypoxic conditions, TGF-β signalling upregulates RBFOX2 in BrC cells (MCF-7 and HCC-1806), thereby inhibiting ESRP1 at the transcriptional level, consequently fostering the pro-metastatic MENAΔ11a subtype, enhancing epithelial-mesenchymal transition (EMT) in BrC cells, and augmenting invasiveness. 101A lower ESRP1/RBFOX2 ratio has been correlated with increased metastatic risk in BrC patients. 82Additionally, RBFOX2 modulates Dicer expression via direct interaction with pre-miR-107, inhibiting its maturation and subsequently increasing the invasiveness of BrC cell lines. 105urthermore, in the more aggressive TNBC, the oncogenic kinase NEK2 stimulates the expression and activity of RBFOX2, thereby promoting the migratory and invasive phenotypes of TNBC cell lines (MDA-MB-231). 7n various other cancer types, RBFOX2 has displayed similar tumour-promoting functions.For instance, in AML mouse bone marrow cells (MLL-AF9) and PDX models, Rbfox2 inhibits bone marrow stem cell differentiation. 29In in vitro cultures of GC cell lines (SGC7901 and MGC803), RBFOX2 overexpression has been shown to enhance the malignant characteristics of GC cells by activating the PI3K/AKT and MAPK signalling pathways. 96Another study conducted with GC cell lines (AGS and MKN28) revealed that LINC00893 directly binds to RBFOX2, inducing its ubiquitination and degradation.This interaction led to the inhibition of proliferation, migration and invasion of GC cells. 102dditionally, in a cell-derived xenograft (CDX) model constructed from MKN45, a GC cell line derived from the gastric lymph nodes of female signet ring cell carcinoma patients, RBFOX2 was found to promote tumour growth and metastasis. 102These findings underscore the multifaceted role of RBFOX2 in GC progression, shedding light on potential therapeutic avenues.In CRC cells (HCT116), RBFOX2 directly binds to ARHGEF7 pre-mRNA, promoting the inclusion of cancer-associated micro-exons and promoting tumour invasion and migration. 99In NPC cells (S26 and 5-8F), RBFOX2 boosts the expression of GOLIM4-L by promoting the inclusion of GOLIM4 exon 7, subsequently enhancing the proliferation and migration of NPC cells in vitro and facilitating CDX model growth in vivo. 32In laryngeal cancer cells (Tu177 and Hep2), the upregulated lncRNA ZFAS1 binds to and upregulates RBFOX2 expression.Elevated RBFOX2 promotes the skipping of MENA exon 11a, ultimately stimulating proliferation, EMT, and invasion of laryngeal cancer cells. 46n ovarian cancer, the lncRNA MALAT1 has been identified to enhance the expression of RBFOX2, subsequently suppressing apoptosis in ovarian cancer cells (HEY and OVARY1847). 104Moreover, in the Ewing's sarcoma cell line (A673), RBFOX2 has been found to bind to ADD3 (a phenotypic driver of Ewing's sarcoma) pre-mRNA.This interaction inhibits the inclusion of ADD3 exon 14, promoting the mesenchymal phenotype of Ewing's sarcoma cells. 44These insights illuminate the intricate regulatory roles of RBFOX2 in different cancer contexts, providing valuable clues for targeted therapeutic interventions.
In contrast to these findings, some studies reveal that RBFOX2 can also exert inhibitory effects on tumour proliferation and invasion.For example, CD47, a receptor widely expressed in cancer cells facilitating evasion from immune system phagocytosis, experiences downregulation of splice isoforms through RBFOX2 in paediatric AML-derived HSCs/HPCs, subsequently impeding tumour proliferation. 6In PDAC cells (BxPC3), RBFOX2 has been identified as a promoter of MPRIP exon 23 inclusion.This inclusion enhances the capacity of MPRIP to inhibit RHOA through its RHO GTPase activating protein function, thereby suppressing tumour cell invasion, migration and in vivo metastasis as demonstrated in a PDX model. 51Recent studies have found that in PDAC, downregulation of RBFOX2 can promote skipping of ABI1 exon 9, causing ABI1 to redistribute from the cytoplasm to the cell periphery.ABI1 at the periphery participates in assembling the WAVE signalling complex, promoting actin polymerisation and cytoskeleton remodeling by activating the Arp2/3 complex, thereby enhancing cell migration and invasion, and causing chemotherapy resistance. 100,109These findings underscore the significant impact of RBFOX2-mediated splicing regulation on the invasive behaviour of PDAC cells, providing potential avenues for therapeutic intervention.
Moreover, in GBM cells (U87MG), RBFOX2 counters SON by promoting PTBP2 exon 10 inclusion, resulting in inhibition of GBM cell proliferation and maintenance of tumour stem cells. 43K63-mediated ubiquitination is a specific type of protein modification process that typically occurs at the Lys63 site of the protein. 110Unlike other types of ubiquitination (such as K48 ubiquitination), K63-linked ubiquitination usually does not lead to the degradation of the modified protein but forms a persistent modification, playing a regulatory role in the cell. 111Notably, the latest study by Li et al. explains a new role of RBFOX2 in GBM and highlights a novel phenomenon of K63-mediated ubiquitination.Specifically, in GBM, the upregulation of FBXO7 can dimethylate Arg341 and Arg441 of RBFOX2 through PRMT5, followed by K63-linked ubiquitination at Lys249.This modification enhances RBFOX2 stability and upregulation.The upregulated RBFOX2 promotes the inclusion of FoxM1 exon Va, making FoxM1 more susceptible to MEK1 phosphorylation, thereby increasing the levels of CD44, CD9 and ID1, ultimately leading to GBM stem cell self-renewal and mesenchymal transformation. 103Targeting the FBXO7-RBFOX2 axis could become a potential strategy for GBM treatment.In malignant gliomas, RBFOX2 acts as a reader, recognising hm5C introduced by NSUN5 and TET2 on CTNNB1 caRNA, leading to CTNNB1 caRNA degradation.CTNNB1 encodes the β-catenin protein.This process inhibits the βcatenin signalling pathway, blocking the phagocytosis of tumour-associated macrophages and resulting in strong immune evasion in malignant gliomas. 30ascinatingly, in endometrial cancer cell lines (KLE and Ishikawa), circRAPGEF5 has been found to competitively bind to the CTD of RBFOX2, specifically residues 266−367.This interaction prevents RBFOX2 from binding to TFRC pre-mRNA, showcasing the intricate regulatory network governing RNA splicing in cancer cells.This event hinders the inclusion of TFRC exon 4, rendering ferroptosis resistance in endometrial cancer cells. 45

RBFOX2-mediated splicing regulation in anticancer drug resistance
The emergence of drug resistance in tumour cells poses a significant obstacle to the effectiveness of chemotherapy and contributes to unfavourable prognoses. 112Recent studies have uncovered the pivotal role of stabilising the splicing activity of RBFOX2 in conferring resistance to anticancer therapies. 6AM, a widely used selective estrogen receptor modulator, plays a critical role in BrC treatment. 113Notably, statistics reveal that 2 years of TAM therapy can provide long-term therapeutic benefits lasting up to 20 years for premenopausal BrC patients with estrogen receptorpositive (ER + ) tumours. 114RBFOX2's role in interfering with the transcriptional activity of steroid receptors, including the modulation of ligands like TAM, was first reported in 2002. 11The RBFOX2-DN isoform, a major negative regulator of RBFOX2's canonical splicing activity, may potentially underlie resistance to other steroid receptor modulators such as Raloxifene, 115 Fulvestrant 116 and Antiandrogens 117 in ER + BrC. 118ebecsinib, a small molecule exhibiting the ability to counteract aberrant gene overediting prompted by malignancies, presents a hopeful avenue for addressing drug resistance and relapse in patients with high-risk myelofibrosis and secondary AML driven by leukaemia stem cells. 119In cord-blood-derived hematopoietic stem and progenitor cells, downregulation-induced splicing dysregulation of RBFOX2 sensitises cells to Rebecsinib treatment, as demonstrated in both colony survival and colony replating assays. 6

TARGETING RBFOX2: UNRAVELING THERAPEUTIC STRATEGIES FOR DISEASE MODULATION
RBFOX2, with its multifaceted role in alternative splicing, emerges as a crucial player in the pathogenesis of CVDs, cancers and other disorders, notably contributing to treatment resistance in malignant tumours.Consequently, targeting RBFOX2 holds significant promise as a cancer therapy.Despite this potential, there is a conspicuous absence of reports on drugs specifically designed to target RBFOX2.Traditionally, RBPs have posed challenges as drug targets. 120However, recent advancements in protein degradation techniques, such as proteolysis-targeting chimeras (PROTAC), have opened new avenues (Figure 5). 121RNA-PROTAC, comprising a genetically encoded RNA scaffold and a synthetic bifunctional small molecule, orchestrates the degradation of target proteins through ubiquitin-proteasome-mediated pathways. 122Studies indicate that Aptamer-based RNA-PROTAC effectively induces the degradation of RBFOX1, a close homologue of RBFOX2, by targeting the specific RBFOX1 binding sequence (-UGCAUGU-). 123Given the structural similarity between RBFOX1 and RBFOX2 in their binding sequences (-UGCAUG-), Aptamer-based RNA-PROTAC presents a promising avenue for RBFOX2 degradation.
Additionally, targeting upstream genes that influence RBFOX2 expression provides an alternative strategy.Notably, NEK2, a cancer-associated factor linked to tumour progression and treatment resistance, promotes the premesenchymal splicing pattern of TNBC cells by upregulating RBFOX2 expression. 7Despite the therapeutic potential of NEK2, no clinically approved NEK2-targeting drugs exist.T-1101 tosylate, a synthetic compound, induces NEK2 degradation and has shown anti-tumour activity in HCC and BrC models. 124Promisingly, T-1101 tosylate has progressed to phase I clinical trials as an orally F I G U R E 5 RNA-PROTAC: targeted degradation strategy extending to RBFOX proteins.RNA-PROTAC consists of a genetically encoded RNA scaffold (containing an aptamer, linker and a RNA consensus binding element [RCBE]) and a synthetic bifunctional small molecule (one end binds the RNA scaffold and the other end binds E3 ubiquitin ligase).When the RCBE of RNA-PROTAC contains the RNA-binding fox-1 homologue 1 (RBFOX1) binding sequence (UGCAUGU), RBFOX1 can bind to the RNA scaffold of the RNA-PROTAC.At the same time, the bifunctional small molecules on RNA-PROTAC can recruit E3 ubiquitin ligase to the RNA scaffold in a non-covalent manner.The recruited E3 ubiquitin ligase specifically induces the ubiquitination of the target protein (i.e., RBFOX1) and its degradation through the ubiquitin-proteasome pathway.This targeting method is also expected to be extended to RBFOX2.PROTAC, proteolysis targeting chimera; RCBE, RNA consensus binding element; Ub, ubiquitin.Created with BioRender.com.administered anticancer agent. 124Moreover, imidazo pyridine derivatives, including MBM-55 and 28e, have exhibited potent inhibitory effects on NEK2. 124These compounds inhibit NEK2 activity by forming conserved hydrogen bonds, effectively impeding the proliferation of diverse tumour cells, including those in BrC, HCC and CRC. 125,126s research into RBFOX2-related diseases deepens, the development of therapies targeting RBFOX2 becomes increasingly urgent and promising.Currently, RBFOX2targeted research remains exploratory, with no publicised outcomes.However, with ongoing research, it is anticipated that RBFOX2-targeted therapies will progress to clinical applications, offering improved prognoses for patients in need.

CONCLUDING REMARKS
Alternative splicing of RNA is pivotal for modulating various isoforms of downstream genes.Among RBPs, the RBFOX family, particularly RBFOX2, plays a crucial role in alternative RNA splicing, impacting diseases such as cancer and heart disease.RBFOX2 has emerged as a diagnostic marker in cancer and a potential therapeutic target.Moving forward, RBFOX2 assumes a multifaceted role extending beyond mere disease involvement.Notably, it plays a crucial part in growth, development and maintenance of bodily homeostasis by modulating tissue-specific alternative splicing. 127For instance, Rbfox2 neural crest-specific deletion mice, constructed using the Cre-LoxP recombinase system, develop craniofacial malformations such as cleft palate due to dysregulation of the Rbfox2-TGF-β-Tak1 signalling axis during development.This suggests that RBFOX2 expression defects are key predisposing factors for neural crest developmental defects. 128Moreover, in the murine liver, Rbfox2 orchestrates cholesterol homeostasis by governing the alternative splicing of Scarb1.These findings underscore the intricate regulatory functions of RBFOX2 in diverse physiological processes. 52lthough RBFOX2 expression disorders have been found in various diseases, the limitations of selected experimental analysis methods and research entry points mean that most results only indicate a statistical association between RBFOX2 and disease development.The causal relationship between RBFOX2 dysregulation and disease progression cannot yet be confirmed.Currently, we can only assert that RBFOX2 disorders are key predisposing factors for HLHS and neural crest developmental defects.Future studies could involve functional experiments in the laboratory, such as gene editing technologies (meganuclease, zinc-finger nuclease [ZFN], transcription activatorlike effector nuclease [TALEN] or CRISPR/Cas9), to artificially regulate the target gene and observe its effects on cells or model animals.Fully exploring the causal relationship between RBFOX2 dysregulation and disease development will greatly enhance our understanding of disease pathogenesis and aid in developing new treatments and preventive measures.
Current basic research has unveiled the initial understanding of how RBFOX2 regulates alternative RNA splicing.Despite this progress, the full-length structure of RBFOX family proteins remains elusive, limiting indepth exploration.Future studies employing advanced techniques such as nuclear magnetic resonance or cryoelectron microscopy are poised to reveal the complete molecular structure of RBFOX2, expanding our comprehension significantly.
Protein post-translational modifications (PTMs), including potential phosphorylation and methylation sites within RBFOX2, are areas ripe for investigation.These PTMs likely influence RBFOX2 function and disease development.Additionally, RBFOX2 exhibits multiple isoforms regulated by alternative splicing.While existing research predominantly focuses on overall RBFOX2 expression, understanding specific isoform expression could resolve the current controversies.Future exploration of PTMs and RBFOX2 isoforms promises a comprehensive understanding of its role in cancer and other diseases.
Given its diverse roles, particularly in malignancies, targeting RBFOX2 emerges as a pivotal strategy for translating research into clinical applications.Innovative technologies such as RNA-PROTAC and NEK2 inhibitors (such as T-1101 tosylate, MBM-55 and 28e) offer promising avenues for targeted degradation and reduction of RBFOX2 levels.With ongoing advancements, RBFOX2-related research is poised to enhance diagnostic accuracy, prognostic prediction and therapeutic efficacy in the near future.

A U T H O R C O N T R I B U T I O N S
Conceptualisation, writing-original draft and visualisation: Jinze Shen.Writing-original draft and visualisation: Jianqiao Shentu.Visualisation; Chenming Zhong and Qiankai Huang.Conceptualisation, writing-review and editing and funding acquisition: Shiwei Duan.

A C K N O W L E D G E M E N T S
The authors would like to express their sincere gratitude for the support provided by the Qiantang Scholars Fund in Hangzhou City University (grant no.210000-581835) for this study.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare they have no conflicts of interest.

E T H I C S S TAT E M E N T Not applicable.
O R C I D Shiwei Duan https://orcid.org/0000-0001-7682-2877

F I G U R E 2
RNA-binding fox-1 homologue 2 (RBFOX2) splicing modes: direct and indirect binding mechanisms unraveled.

F I G U R E 3
RNA-binding fox-1 homologue 2 (RBFOX2): orchestrating cardiomyocyte function.RBFOX2 plays a pivotal role in regulating physiological mechanisms closely associated with cardiomyocyte function, such as excitation-contraction coupling (E-C coupling), calcium ion channels and mitochondrial metabolism, by influencing the alternative splicing of downstream genes.Dysregulation of RBFOX2 has been implicated in the onset of various cardiovascular diseases, including arrhythmias and cardiomyopathy.Specifically, RBFOX2 can promote the expression of Jph2 by inhibiting the maturation of miR-34a, thereby enhancing the conduction of electrical signals in cardiomyocytes.RBFOX2 also inhibits the inclusion of Cacna1c exon 9′, thereby stabilising the number of L-type calcium channels in cardiomyocyte membranes and promoting calcium transients after depolarisation.Additionally, RBFOX2 promotes the inclusion of MICU1 exon 5′, enhancing mitochondrial Ca 2+ uptake and thereby boosting energy metabolism.RBFOX2 also facilitates the inclusion of Enah exon 13, Sorbs2 exon 4 and Tpm1 exon 9b, while inhibiting the inclusion of Tpm1 exon 6a, thus promoting muscle fibre contraction.EI, exon inclusion; EMG, electromyographic; ES, exon skipping.Created with BioRender.com.